Adenoviruses are a large family of double-stranded DNA viruses that replicate in the nucleus of the host cell. The viral genes are categorized as either "early" or "late" genes. These temporal categories are based on when the genes are transcribed into mRNA during the virus life cycle. Transcription occurs coordinately and the transition from early to late transcription occurs at approximately 10 hours post-infection, coinciding with DNA replication. As the viral genes are expressed, there is a gradual reduction in host cell RNA, DNA and protein synthesis while the quantity of viral proteins and nucleic acids slowly rises. By about 36 hours post-infection, the host cell disintegrates and the virus is released into the environment.
Adenoviruses are thus ideal candidates for generating vectors useful in gene therapy because the virus uses the host cell's own machinery to synthesize viral RNA, DNA and proteins. Furthermore, the transcription of the adenovirus genes, the organization of the genome and the DNA sequence of the genome have been well defined. Thus, non-viral DNA encoding proteins of interest can be inserted into the adenovirus genome at appropriate locations, and these proteins can be readily expressed in the host cell.
Because adenovirus replication ultimately results in cell death, previous adenovirus vectors were designed to reduce virus replication. Reduced viral replication has been accomplished by deleting or mutating portions of early genes such as the E1a/E1b region, as this region of the genome regulates the expression of various other adenovirus genes required for DNA replication. Berkner, K. L., Biotechniques 6:616-629 (1988). Horwitz, M. S., "Virology," 2d ed., Raven Press Ltd., p.1679-1720 (1990).
Although E1a/E1b-deleted adenoviruses exhibit reduced virus replication, these vectors are inefficiently packaged into the viral capsid due to the large genome generated by the additional transgene DNA. The inefficient packaging reduces the titer of the virus stocks by 2-3 logs in comparison to traditional vectors. The low titer of the E1a/E1b-deleted viruses reduces their usefulness, especially for applications to entire organs such as the lung.
Vectors have been constructed with an additional deletion in the E3 region. This deletion increases the amount of non-viral DNA that can be inserted into the vector while maintaining efficient packaging of the recombinant virus. Engelhardt, J. F. et al., Nature Genet. 4:27-34 (1993). However, there is speculation that expression of the E3 gene aids virus-infected cells in avoiding the immune response of the host. Therefore, deleting the E3 region is undesirable, as the lack of E3 protein expression increases the chance that the virus infected cells will be rejected by the immune system of the host.
E3-inclusive, E1a/E1b-deleted adenovirus vectors currently exist and have been approved for clinical trial. A major disadvantage of these vectors, however, is again, inefficient packaging because of large genome size that leads to much lower titers than traditional vectors, making them less useful in large scale human applications. With deletions in other regions of the adenovirus genome, the E3 region could be retained and the appropriate viral genome size could be achieved for the production of high titer stocks for clinical use.
A particularly useful application for adenovirus vectors is in the treatment of cystic fibrosis (CF) by gene therapy. Various gene therapy approaches have been considered for cystic fibrosis without sufficient results. One such approach is to selectively reconstitute cystic fibrosis transmembrane regulator (CFTR) gene expression in the surface epithelium using gene transfer substrates delivered directly into the airway. Although transfection of airway epithelial cells has been achieved in vivo with cationic liposomes, efficiencies have been below what is required for therapeutic efficacy. Hazinski, T. A. et al., Am. J. Respir. Cell. Mol. Biol. 4:206-209 (1991); Yoshimura, K. et al., Nucleic Acids Res. 20:3233-3240 (1992). Likewise, recombinant retroviruses carrying the CFTR gene have been unacceptable because efficient and stable recombinant gene expression can be accomplished in proximal airway with recombinant retroviruses only if the epithelium is undifferentiated and regenerating at the time of exposure to virus, a situation that is difficult to simulate in patients. Engelhardt, J. E. et al., J. Clin. Invest. 90:2598-2607 (1992).
The use of recombinant adenoviruses for cystic fibrosis gene therapy is thus particularly attractive especially considering the important advantages of adenovirus vectors, including their natural tropicity to human airway, growth to extremely high titers, and their ability to transfer and express recombinant genes in nondividing cells. Graham, F. L. et al., "Gene Transfer and Expression Protocol," E. J. Murray ed., The Human Press, Inc., Clifton, N.J. 109-128 (1992). Due to these advantages, recombinant adenoviruses have been used to transfer genes for .alpha.-1-antitrypsin and CFTR into lungs of cotton rats. Rosenfeld, M. A. et al., Science 252:431-434 (1991 ); Rosenfeld, M. A. et al., Cell 68:143-155 (1992).
It would thus be desirable to produce an improved recombinant adenovirus vector. It would also be desirable to produce a recombinant adenovirus vector which has a deletion of at least a portion of the E1region, retains at least a portion of the E3 region and contains an additional deletion to accommodate a transgene and/or other mutations which result in reduced expression or over-expression of adenoviral protein and/or reduced viral replication. It would further be desirable to produce a recombinant adenovirus vector which has a deletion of at least a portion of the E1 region, retains at least a portion of the E3 region, contains an additional deletion to accommodate a transgene and/or other mutations which result in reduced expression or over-expression of adenoviral protein and/or reduced viral replication, and which contains a transgene of interest, for example, the CFTR gene. In addition, it would be desirable to produce a gene expression system useful for the study and employment of therapeutic approaches.